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1.
BMC Res Notes ; 13(1): 14, 2020 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-31910899

RESUMO

OBJECTIVE: F-spondin is part of a group of evolutionarily conserved extracellular matrix proteins in vertebrates. It is highly expressed in the embryonic floor plate, and it can bind to the ECM and promote neuronal outgrowth. A characterization of F-spondin expression patterns in the adult zebrafish brain was previously reported by our group. However, given its importance during development, we aimed to obtain a detailed description of green fluorescent protein (GFP) expression driven by the spon1b promotor, in the developing zebrafish brain of the transgenic Tg(spon1b:GFP) line, using light sheet fluorescence microscopy (LSFM). RESULTS: Images obtained in live embryos from 22 to 96 h post fertilization confirmed our earlier reports on the presence of spon1b:GFP expressing cells in the telencephalon and diencephalon (olfactory bulbs, habenula, optic tectum, nuclei of the medial longitudinal fasciculus), and revealed new spon1b:GFP populations in the pituitary anlage, dorso-rostral cluster, and ventro-rostral cluster. LSFM made it possible to follow the dynamics of cellular migration patterns during development. CONCLUSIONS: spon1b:GFP larval expression patterns starts in early development in specific neuronal structures of the developing brain associated with sensory-motor modulation. LSFM evaluation of the transgenic Tg(spon1b:GFP) line provides an effective approach to characterize GFP expression patterns in vivo.


Assuntos
Encéfalo/embriologia , Encéfalo/metabolismo , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimento , Animais , Embrião não Mamífero/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fertilização , Proteínas de Fluorescência Verde/metabolismo , Habenula/embriologia , Habenula/metabolismo , Proteínas de Peixe-Zebra/metabolismo
2.
Biol Open ; 8(5)2019 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-31085547

RESUMO

Glioblastoma (GBM) is the most prevalent type of primary brain tumor. Treatment options include maximal surgical resection and drug-radiotherapy combination. However, patient prognosis remains very poor, prompting the search for new models for drug discovery and testing, especially those that allow assessment of in vivo responses to treatment. Zebrafish xenograft models have an enormous potential to study tumor behavior, proliferation and cellular interactions. Here, an in vivo imaging and proliferation assessment method of human GBM xenograft in zebrafish larvae is introduced. Zebrafish larvae microinjected with fluorescently labeled human GBM cells were screened daily using a stereomicroscope and imaged by light sheet fluorescence microscopy (LSFM); volumetric modeling and composite reconstructions were done in single individuals. Larvae containing tumors were enzymatically dissociated, and proliferation of cancer cells was measured using dye dilution by flow cytometry. GBM micro-tumors formed mainly in the zebrafish yolk sac and perivitelline space following injection in the yolk sac, with an engraftment rate of 73%. Daily image analysis suggested cellular division, as micro-tumors progressively grew with differentiated fluorescence intensity signals. Using dye dilution assay by flow cytometry, at least three GBM cells' division cycles were identified. The combination of LSFM and flow cytometry allows assessment of proliferation and tumor growth of human GBM inside zebrafish, making it a useful model to identify effective anti-proliferative agents in a preclinical setting.

3.
Front Oncol ; 9: 245, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31024847

RESUMO

Acute leukemia is a heterogeneous set of diseases affecting children and adults. Current prognostic factors are not accurate predictors of the clinical outcome of adult patients and the stratification of risk groups remains insufficient. For that reason, this study proposes a multifactorial analysis which integrates clinical parameters, ex vivo tumor characterization and behavioral in vivo analysis in zebrafish. This model represents a new approach to understand leukemic primary cells behavior and features associated with aggressiveness and metastatic potential. Xenotransplantation of primary samples from patients newly diagnosed with acute leukemia in zebrafish embryos at 48 hpf was used to asses survival rate, dissemination pattern, and metastatic potential. Seven samples from young adults classified in adverse, favorable or intermediate risk group were characterized. Tumor heterogeneity defined by Leukemic stem cell (LSC) proportion, was performed by metabolic and cell membrane biomarkers characterization. Thus, our work combines all these parameters with a robust quantification strategy that provides important information about leukemia biology, their relationship with specific niches and the existent inter and intra-tumor heterogeneity in acute leukemia. In regard to prognostic factors, leukemic stem cell proportion and Patient-derived xenografts (PDX) migration into zebrafish were the variables with highest weights for the prediction analysis. Higher ALDH activity, less differentiated cells and a broader and random migration pattern are related with worse clinical outcome after induction chemotherapy. This model also recapitulates multiple aspects of human acute leukemia and therefore is a promising tool to be employed not only for preclinical studies but also supposes a new tool with a higher resolution compared to traditional methods for an accurate stratification of patients into worse or favorable clinical outcome.

4.
Anat Sci Educ ; 11(2): 137-145, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28759705

RESUMO

Visualizing anatomical structures and functional processes in three dimensions (3D) are important skills for medical students. However, contemplating 3D structures mentally and interpreting biomedical images can be challenging. This study examines the impact of a new pedagogical approach to teaching neuroanatomy, specifically how building a 3D-model from oil-based modeling clay affects learners' understanding of periventricular structures of the brain among undergraduate medical students in Colombia. Students were provided with an instructional video before building the models of the structures, and thereafter took a computer-based quiz. They then brought their clay models to class where they answered questions about the structures via interactive response cards. Their knowledge of periventricular structures was assessed with a paper-based quiz. Afterward, a focus group was conducted and a survey was distributed to understand students' perceptions of the activity, as well as the impact of the intervention on their understanding of anatomical structures in 3D. Quiz scores of students that constructed the models were significantly higher than those taught the material in a more traditional manner (P < 0.05). Moreover, the modeling activity reduced time spent studying the topic and increased understanding of spatial relationships between structures in the brain. The results demonstrated a significant difference between genders in their self-perception of their ability to contemplate and rotate structures mentally (P < 0.05). The study demonstrated that the construction of 3D clay models in combination with autonomous learning activities was a valuable and efficient learning tool in the anatomy course, and that additional models could be designed to promote deeper learning of other neuroanatomy topics. Anat Sci Educ 11: 137-145. © 2017 American Association of Anatomists.


Assuntos
Educação de Graduação em Medicina/métodos , Modelos Anatômicos , Neuroanatomia/educação , Aprendizagem Baseada em Problemas/métodos , Adulto , Silicatos de Alumínio , Encéfalo/anatomia & histologia , Argila , Colômbia , Computadores , Currículo , Avaliação Educacional/estatística & dados numéricos , Feminino , Grupos Focais , Humanos , Masculino , Avaliação de Programas e Projetos de Saúde , Autorrelato , Fatores Sexuais , Estudantes de Medicina/estatística & dados numéricos , Adulto Jovem
5.
Rev. colomb. cancerol ; 21(4): 212-224, oct.-dic. 2017. tab, graf
Artigo em Espanhol | LILACS | ID: biblio-900477

RESUMO

Resumen El pez cebra es un modelo establecido para el estudio del desarrollo en vertebrados y es especialmente útil para la investigación del proceso de hematopoyesis y las enfermedades asociadas a esta. Los linajes principales, los genes y los procesos de desarrollo con los seres humanos son conservados. En los últimos años, el pez cebra se ha utilizado cada vez más como un modelo para estudiar enfermedades hematopoyéticas humanas, incluyendo la leucemia linfoblástica aguda. Esta revisión evidencia la importancia del estudio de esta enfermedad en Colombia debido a las diferencias de la etiología que presenta este tipo de leucemia en comparación con otros países. Además, describe la aplicación del pez cebra como una herramienta alternativa para investigaciones preclínicas de la leucemia linfoblástica aguda. Este modelo es asequible, facilita la experimentación, su manipulación es relativamente simple y tiene gran versatilidad para estudios moleculares y genéticos del cáncer y está disponible en Colombia.


Abstract The zebrafish is an established model for the study of vertebrate development, and it is specially useful for the research into haematopoiesis and diseases associated with this process. Major lineages, genes, and developmental processes are conserved between zebrafish and humans. Thus it has been increasingly used as a model for a number of haematopoietic human diseases, such as acute lymphoblastic leukaemia. This review highlights the importance of the study of this disease in Colombia, because of the differences in its aetiology compared to other countries. It also describes the application of the zebrafish as an alternative tool for pre-clinical research of acute lymphoblastic leukaemia. This model is affordable, facilitates experimentation and handling, and is extremely versatile for molecular and genetic studies into cancer, and it is now available in Colombia.


Assuntos
Humanos , Peixe-Zebra , Modelos Animais , Leucemia-Linfoma Linfoblástico de Células Precursoras , Leucemia
6.
J Vis Exp ; (127)2017 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-28994774

RESUMO

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique's relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.


Assuntos
Doença de Chagas/diagnóstico , Trypanosoma cruzi/patogenicidade , Animais , Movimento Celular , Modelos Animais de Doenças , Humanos , Larva , Peixe-Zebra
7.
J Neurosci ; 37(7): 1900-1909, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28087763

RESUMO

The circadian system may regulate adult neurogenesis via intracellular molecular clock mechanisms or by modifying the environment of neurogenic niches, with daily variation in growth factors or nutrients depending on the animal's diurnal or nocturnal lifestyle. In a diurnal vertebrate, zebrafish, we studied circadian distribution of immunohistochemical markers of the cell division cycle (CDC) in 5 of the 16 neurogenic niches of adult brain, the dorsal telencephalon, habenula, preoptic area, hypothalamus, and cerebellum. We find that common to all niches is the morning initiation of G1/S transition and daytime S-phase progression, overnight increase in G2/M, and cycle completion by late night. This is supported by the timing of gene expression for critical cell cycle regulators cyclins D, A2, and B2 and cyclin-dependent kinase inhibitor p20 in brain tissue. The early-night peak in p20, limiting G1/S transition, and its phase angle with the expression of core clock genes, Clock1 and Per1, are preserved in constant darkness, suggesting intrinsic circadian patterns of cell cycle progression. The statistical modeling of CDC kinetics reveals the significant circadian variation in cell proliferation rates across all of the examined niches, but interniche differences in the magnitude of circadian variation in CDC, S-phase length, phase angle of entrainment to light or clock, and its dispersion. We conclude that, in neurogenic niches of an adult diurnal vertebrate, the circadian modulation of cell cycle progression involves both systemic and niche-specific factors.SIGNIFICANCE STATEMENT This study establishes that in neurogenic niches of an adult diurnal vertebrate, the cell cycle progression displays a robust circadian pattern. Common to neurogenic niches located in diverse brain regions is daytime progression of DNA replication and nighttime mitosis, suggesting systemic regulation. Differences between neurogenic niches in the phase and degree of S-phase entrainment to the clock suggest additional roles for niche-specific regulatory mechanisms. Understanding the circadian regulation of adult neurogenesis can help optimize the timing of therapeutic approaches in patients with brain traumas or neurodegenerative disorders and preserve neural stem cells during cytostatic cancer therapies.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Ritmo Circadiano/fisiologia , Neurogênese/fisiologia , Núcleo Supraquiasmático/fisiologia , Animais , Bromodesoxiuridina/metabolismo , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Ritmo Circadiano/efeitos dos fármacos , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Escuridão , Masculino , Neurogênese/efeitos dos fármacos , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , RNA Mensageiro/metabolismo , Núcleo Supraquiasmático/citologia , Núcleo Supraquiasmático/efeitos dos fármacos , Peixe-Zebra
8.
PLoS One ; 7(6): e37593, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22768035

RESUMO

F-spondin, an extracellular matrix protein, is an important player in embryonic morphogenesis and CNS development, but its presence and role later in life remains largely unknown. We generated a transgenic zebrafish in which GFP is expressed under the control of the F-spondin (spon1b) promoter, and used it in combination with complementary techniques to undertake a detailed characterization of the expression patterns of F-spondin in developing and adult brain and periphery. We found that F-spondin is often associated with structures forming long neuronal tracts, including retinal ganglion cells, the olfactory bulb, the habenula, and the nucleus of the medial longitudinal fasciculus (nMLF). F-spondin expression coincides with zones of adult neurogenesis and is abundant in CSF-contacting secretory neurons, especially those in the hypothalamus. Use of this new transgenic model also revealed F-spondin expression patterns in the peripheral CNS, notably in enteric neurons, and in peripheral tissues involved in active patterning or proliferation in adults, including the endoskeleton of zebrafish fins and the continuously regenerating pharyngeal teeth. Moreover, patterning of the regenerating caudal fin following fin amputation in adult zebrafish was associated with F-spondin expression in the blastema, a proliferative region critical for tissue reconstitution. Together, these findings suggest major roles for F-spondin in the CNS and periphery of the developing and adult vertebrate.


Assuntos
Envelhecimento/genética , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Embrião não Mamífero/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Larva/genética , Larva/crescimento & desenvolvimento , Masculino , Neurogênese/genética , Especificidade de Órgãos/genética , Homologia de Sequência de Aminoácidos , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismo
9.
J Neurotrauma ; 24(2): 354-66, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17375999

RESUMO

Following traumatic brain injury (TBI), the cytoskeletal protein alpha-II-spectrin is proteolyzed by calpain and caspase-3 to signature breakdown products. To determine whether alpha -II-spectrin proteolysis is a potentially reliable biomarker for TBI in humans, the present study (1) examined levels of spectrin breakdown products (SBDPs) in cerebrospinal fluid (CSF) from adults with severe TBI and (2) examined the relationship between these levels, severity of injury, and clinical outcome. This prospective case control study enrolled 41 patients with severe TBI, defined by a Glasgow Coma Scale (GCS) score of < or =8, who underwent intraventricular intracranial pressure monitoring. Patients without TBI requiring CSF drainage for other medical reasons served as controls. Ventricular CSF was sampled from each patient at 6, 12, 24, 48, 72, 96, and 120 h following TBI and analyzed for SBDPs. Outcome was assessed using the Glasgow Outcome Score (GOS) 6 months after injury. Calpain and caspase-3 mediated SBDP levels in CSF were significantly increased in TBI patients at several time points after injury, compared to control subjects. The time course of calpain mediated SBDP150 and SBDP145 differed from that of caspase-3 mediated SBDP120 during the post-injury period examined. Mean SBDP densitometry values measured early after injury correlated with severity of injury, computed tomography (CT) scan findings, and outcome at 6 months post-injury. Taken together, these results support that alpha -II-spectrin breakdown products are potentially useful biomarker of severe TBI in humans. Our data further suggests that both necrotic/oncotic and apoptotic cell death mechanisms are activated in humans following severe TBI, but with a different time course after injury.


Assuntos
Lesões Encefálicas/líquido cefalorraquidiano , Proteínas de Transporte/líquido cefalorraquidiano , Proteínas dos Microfilamentos/líquido cefalorraquidiano , Espectrina/líquido cefalorraquidiano , Adolescente , Adulto , Idoso , Biomarcadores/líquido cefalorraquidiano , Calpaína/líquido cefalorraquidiano , Estudos de Casos e Controles , Caspase 3/líquido cefalorraquidiano , Feminino , Escala de Coma de Glasgow , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos
10.
J Neurochem ; 98(3): 700-12, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16893416

RESUMO

Axonal injury is one of the key features of traumatic brain injury (TBI), yet little is known about the integrity of the myelin sheath. We report that the 21.5 and 18.5-kDa myelin basic protein (MBP) isoforms degrade into N-terminal fragments (of 10 and 8 kDa) in the ipsilateral hippocampus and cortex between 2 h and 3 days after controlled cortical impact (in a rat model of TBI), but exhibit no degradation contralaterally. Using N-terminal microsequencing and mass spectrometry, we identified a novel in vivo MBP cleavage site between Phe114 and Lys115. A MBP C-terminal fragment-specific antibody was then raised and shown to specifically detect MBP fragments in affected brain regions following TBI. In vitro naive brain lysate and purified MBP digestion showed that MBP is sensitive to calpain, producing the characteristic MBP fragments observed in TBI. We hypothesize that TBI-mediated axonal injury causes secondary structural damage to the adjacent myelin membrane, instigating MBP degradation. This could initiate myelin sheath instability and demyelination, which might further promote axonal vulnerability.


Assuntos
Lesões Encefálicas/metabolismo , Calpaína/fisiologia , Proteína Básica da Mielina/metabolismo , Sequência de Aminoácidos , Animais , Lesões Encefálicas/enzimologia , Lesões Encefálicas/genética , Lesões Encefálicas/patologia , Modelos Animais de Doenças , Hidrólise , Masculino , Dados de Sequência Molecular , Peso Molecular , Proteína Básica da Mielina/genética , Peptídeo Hidrolases/farmacologia , Peptídeo Hidrolases/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley
11.
Biochem J ; 394(Pt 3): 715-25, 2006 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-16351572

RESUMO

A major theme of TBI (traumatic brain injury) pathology is the over-activation of multiple proteases. We have previously shown that calpain-1 and -2, and caspase-3 simultaneously produced alphaII-spectrin BDPs (breakdown products) following TBI. In the present study, we attempted to identify a comprehensive set of protease substrates (degradome) for calpains and caspase-3. We further hypothesized that the TBI differential proteome is likely to overlap significantly with the calpain- and caspase-3-degradomes. Using a novel HTPI (high throughput immunoblotting) approach and 1000 monoclonal antibodies (PowerBlottrade mark), we compared rat hippocampal lysates from 4 treatment groups: (i) naïve, (ii) TBI (48 h after controlled cortical impact), (iii) in vitro calpain-2 digestion and (iv) in vitro caspase-3 digestion. In total, we identified 54 and 38 proteins that were vulnerable to calpain-2 and caspase-3 proteolysis respectively. In addition, the expression of 48 proteins was down-regulated following TBI, whereas that of only 9 was up-regulated. Among the proteins down-regulated in TBI, 42 of them overlapped with the calpain-2 and/or caspase-3 degradomes, suggesting that they might be proteolytic targets after TBI. We further confirmed several novel TBI-linked proteolytic substrates, including betaII-spectrin, striatin, synaptotagmin-1, synaptojanin-1 and NSF (N-ethylmaleimide-sensitive fusion protein) by traditional immunoblotting. In summary, we demonstrated that HTPI is a novel and powerful method for studying proteolytic pathways in vivo and in vitro.


Assuntos
Lesões Encefálicas/metabolismo , Calpaína/metabolismo , Caspases/metabolismo , Proteoma/metabolismo , Animais , Caspase 3 , Regulação da Expressão Gênica , Hipocampo/metabolismo , Humanos , Masculino , Proteômica , Ratos , Ratos Sprague-Dawley
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